Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the present invention relates to new oligonucleotide analogs with valuable physical, biological and pharmacological properties and to a process for their preparation.
• Their application relates to their use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), as probes for the detection of nucleic acids and as aids in molecular biology.
• Oligonucleotides are increasingly used as inhibitors of gene expression (G. Zon, Pharmaceutical Research 5, 539 (1988); JS Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press; C.
• Antisense oligonucleotides are nucleic acid fragments whose base sequence is complementary to an mRNA to be inhibited.
• This target mRNA can be of cellular, viral or other pathogenic origin.
• Cellular target sequences that can be used are, for example, those of receptors, enzymes, immunomodulators, ion channels or oncogenes.
• RNA polymerase and transactivator proteins are examples of viral targets.
• RSV Raster Sarcoma Virus
• HSV-1 and -2 Herpes Simplex Virus Type I and II
• HIV Human Immunodeficiency Virus
• influenza viruses This uses oligonucleotides that are complementary to the viral nucleic acid.
• Sense oligonucleotides are designed in their sequence so that, for example, they bind ("capture") nucleic acid-binding proteins or nucleic acid-processing enzymes and thus inhibit their biological activity (Helene, 1990).
• Reverse transcriptase, DNA polymerase and transactivator proteins are examples of viral targets.
• Triplex forming oligonucleotides generally target DNA and form a triple helical structure after binding to them. While antisense oligonucleotides generally inhibit the processing (splicing etc.) of the mRNA or its translation into the protein, triplex forming oligonucleotides inhibit the transcription or replication of the DNA (Helene et al., 1990, Uhlmann and Peyman, 1990) , However, it is also possible to bind single-stranded nucleic acids in a first hybridization with an antisense oligonucleotide to form a double strand, which then forms a triplex structure in a second hybridization with a triplex forming oligonucleotide.
• the antisense and triplex binding regions can either be housed in two separate oligonucleotides or in one oligonucleotide.
• Another application of synthetic oligonucleotides are the so-called ribozymes, which destroy the target RNA due to their ribonuclease activity (JJ Rossi and N. Sarver, TIBTECH 8, 179 (1990).
• ribozymes which destroy the target RNA due to their ribonuclease activity
• nucleic acid fragments with suitable labeling are used as so-called DNA probes or DNA probes for the specific hybridization to a nucleic acid to be detected.
• the specific formation of the new double strand is followed with the aid of the marking, which is preferably not radioactive. In this way, genetic, malignant, viral or other pathogens can be detected.
• oligonucleotides usually takes place in such a way that the phosphate backbone, ribose unit or the nucleobases are changed accordingly (Cohen, 1989; Uhlmann and Peyman, 1990).
• Another frequently used method is the preparation of oligonucleotide-5'-conjugates by reacting the 5'-hydroxy group with corresponding phosphorylation reagents.
• Oligonucleotides that are only modified at the 5 'end have the disadvantage that they are broken down in the serum. On the other hand, if all internucleotide phosphate residues are changed, the properties of the oligonuleotides often change drastically.
• the solubility of the methylphosphonate oligonucleotides in aqueous medium is reduced and the hybridization capacity is reduced.
• Phosphorothioate oligonucleotides have a non-specific effect, so that, for example, homooligomers are also effective against viruses.
• Antisense oligonucleotides generally have a uniform polarity, which usually has an antiparallel character when hybridized to RNA (see Table 1; A). In certain cases, for example in the case of oligonucleotides composed of ⁇ -nucleoside units, the polarity can also be parallel (Table 1; B). Triplex forming oligonucleotides can generally hybridize to double-stranded nucleic acids in a sequence-dependent manner in parallel (Table 1; C) or antiparallel (Table 1; D) orientation with respect to the purine-rich nucleic acid strand.
• the base pairing motifs T • AT, G • GC, C + • GC, G • TA, C Me • GC, A • AT and C Pl • GC are used, in which C + is a protonated cytosine residue, C Me is a 5 -Methyl-cytosine residue, C Pl a pseudoiso-cytosine residue and "•" a Hoogsteen or reverse Hoogsteen base pairing.
• C + is a protonated cytosine residue
• C Me is a 5 -Methyl-cytosine residue
• C Pl a pseudoiso-cytosine residue
• "•" a Hoogsteen or reverse Hoogsteen base pairing.
• the use of these Hoogsteen base pairs is limited to purine-rich regions of the double-stranded nucleic acids.
• oligonucleotides with alternating polarity were produced which are due to a (5'5 ') strand change (Table 1; E) or a (3'3') - strand change can alternate if necessary (Table 1; F), can bind purine-rich regions of the opposite strand (Ono et al., Biochemistry (1991) 30, 9914).
• a strand change while maintaining the polarity is possible if a (3'5 ') spacer is installed.
• oligonucleotides can be produced which contain a 5'5 'spacer (Table 1; K) at the 5' end.
• oligonucleotide analogs both 3 '(2') ends can advantageously contain phosphoryl groups.
• oligonucleotide analogs which the in Table 1 C to I Obey the principles of action shown, generally have one Hydroxy group at the 3 'end, so that they are broken down in the serum, mostly are poorly membrane-permeable and can only be easily derivatized at the 5 'end.
• the task is therefore to use specific oligonucleotide analogs Hybridization properties against single and double stranded nucleic acids, increased serum stability, good solubility and specific effectiveness provide.
• Preferred oligonucleotide analogs of the formulas IA and IB are those in which the base B is in the ⁇ position, the nucleotides are in the D configuration, R 2 is in the 2 'position and a is oxy.
• oligonucleotide analogs of the formulas IA and IB are particularly preferred, in which V, Y 'and Y have the meaning of oxy. Also particularly preferred are oligonucleotide analogs of the formula IA and IB, in which V, Y, Y 'and W have the meaning of oxy or oxo. Oligonucleotide analogs of the formulas IA and IB, in which V, Y, Y ', W and U have the meaning of oxy, oxo or hydroxy, are very particularly preferred. Furthermore, oligonucleotide analogs of the formulas IA and IB are preferred, in which R 1 represents hydrogen.
• oligonucleotide analogs of the formula IA and IB in which U, V, W, X, Y 'and Y have the meaning of oxy, oxo or hydroxyl and R 1 is hydrogen.
• oligonucleotides of the formula IA in which r is zero, V is Y 'and R 1 has the meaning of formula II.
• the recurring residues such as R 2 , B, a, d, e, f, g, g ', h, W, V, Y, Y', U, R 3 , R 4 , p, q, G and Z can independently the same or different
• ie V means independently of one another oxy, sulfanediyl or imino.
• Halogen is preferably fluorine, chlorine or bromine.
• Heteroaryl is understood to mean the remainder of a monocyclic or bicyclic (C 3 -C 9 ) heteroaromatic which contains one or two N atoms and / or an S or an O atom in the ring system.
• Typical marker groups are: Oligonucleotide analogs which bind or intercalate and / or cleave or crosslink to nucleic acids contain e.g. B. acridine, psoralen, phenanthridine, naphthoquinone, daunomycin or chloroethylaminoaryl conjugates.
• Typical intercalating and cross-linking residues are:
• Examples of groups NR 3 R 4 in which R 3 and R 4 together with the nitrogen atom carrying them form a 5- to 6-membered heterocyclic ring which additionally contains a further heteroatom are the morpholinyl and the imidazolidinyl radical ,
• the invention is not based on ⁇ - and ⁇ -D- or L-ribofuranosides, ⁇ - and ⁇ -D- or L-deoxyribofuranosides and corresponding five-ring carbocyclic analogues limited, but also applies to oligonucleotide analogs that come from others Sugar building blocks are built, for example ring-expanded and ring-narrowed Sugar, acyclic or other suitable sugar derivatives.
• the invention is furthermore not to the derivatives of the examples listed in formula IA and IB Limited phosphate residue, but also refers to the known Dephospho derivatives.
• Oligonucleotide analogs of the formula IA have one or more 5'5 'spacers (5'5'S) and optionally also one or more 3'3'-spacers (3'3'S) or 2'2'-spacers, which each cause a change in polarity.
• Oligonucleotide analogs of Formula IB have a 3'5 'spacer (3'5'S) or (2'5'S) that does not have the polarity influenced, but for example a refolding of the oligonucleotide analog or a strand change allowed.
• S propane-1,3-diol phosphate, 2-benzyl and 2-octadecyl-oxypropane-1,3-diol phosphates, triethylene glycol or hexaethylene glycol phosphates, which can also be repeated if necessary.
• Nucleotide analogs without a heterocyclic base and phenylene dialkylene residues are further preferred embodiments of S, in particular of (3'3'S). In general, (5'5'S) is longer than (3'3'S) for topological reasons.
• (5'5'S) mostly has 20 to 45, preferably 24 to 36 unbranched bonds, while (3'3'S) has only 5 to 15, preferably 6 to 10 unbranched bonds, provided that a change of strand is sought.
• S is used for refolding the oligonucleotide analog, for example for triplex formation on the single nucleic acid strand, lengths of S corresponding to 2 to 8, preferably 4 to 5, nucleotide units are advantageous.
• Penta- (2-benzyloxy-1,3-propanediol) hexaphosphate and hexa- (propane-1,3-diol) pentaphosphate may be mentioned here as examples, each of which can bring about a (5'5'S) or (3'5'S) linkage ,
• oligonucleotide analogs of the formulas IA and B is carried out similarly to the synthesis of biological oligonucleotides in solution or preferably in solid Phase, if necessary with the help of an automatic synthesizer.
• Solid phase synthesis of oligonucleotides with a phosphate or Phosphate ester residue at the 3 'end is according to the standard phosphoramidite chemistry according to Caruthers (M.D. Matteucci and M.H. Caruthers, J. Am. Chem. Soc. 103, 3185 (1981)) not possible because the first nucleotide building block is via the 3'-hydroxy group is bound to the solid support and therefore from this Syntheses always result in oligonucleotides with a 3'-hydroxy group.
• It different methods according to the solid phase method have been described but all are cumbersome and often do not have derivatives such as phosphate esters or have alkylphosphonates prepared (R.
• the linker arm A which connects the solid support T with the sulfur-containing residue by chemical bonding (amide, ester, etc.) (Damka et al., Nucleic Acids Res. 18, 3813 (1990)), is preferably a succinic acid residue (OC (O) -CH 2 CH 2 -C (O) -), an oxalic acid residue, (OC (O) -C (O) -), an alkylamine, preferably LCAA (long chain alkyl amine), or polyethylene glycol.
• a succinic acid residue is particularly preferred. in certain cases, for example in combination with substituents that cannot withstand prolonged ammonia treatment, more unstable linkers such as the oxalyl linker are advantageous.
• the preparation of solid supports of the formulas IV ac is described in Example 1.
• Preferred bis-amidites are those of the formula Vla
• the introduction of the group Z can also be carried out according to the H-phosphonate method, by first reacting a nucleoside phosphonate of the formula XI wherein R, V, a, B ', Y', X 'and W have the meaning given above, H-phosphonate diesters of the formula VII' formed with a support of the formula IV ' is subjected to an oxidative phosphoramidation (B. Froehler, Tetrahedron Lett. 27, 5575 (1986). In this way, for example, an oligonucleotide with a 3'-terminal cholesteryl group can be produced with cholesteryl-oxycarbonyl-aminoalkylamine in the presence of carbon tetrachloride.
• a (3'5'S) loop can be made up of multiple, preferably 4 to Introduce 5-fold successive coupling with the block of formula XIVb. After that the synthesis is continued in the 3'5 'direction as previously described.
• nucleic acids preferably DNA
• 5'5 'spacers of greater length.
• a Triethylene glycol diphosphate on a chain built in 3'5 'direction or several times, preferably twice by coupling twice with the building block Formula XIVa can be installed at the desired location.
• the Polarity is then synthesized using nucleotide building blocks of formula XIII 5'3 'direction continued.
• a 3'3 'spacer is inserted at the intended location and then the Synthesis of the chain in the 3'5 'direction by condensation with Nucleoside 3'-phosphoramidites continued. It is a 3'3 'spacer
• the introduction of a propane-1,3-diol diphosphate group can be introduced by coupling with the block of formula XIVd.
• the type of the amino protective groups of the bases B 'and the nature of the lower arm A depend in individual cases on the type of the substituent Z, since these must be easily cleavable after complete synthesis.
• Z OiC 3 H 7
• PAC labile phenoxyacetyl
• conjugates have additional functional groups which must be appropriately protected before incorporation into the monomeric building blocks of the formulas V and XIII.
• the carboxyl group of fluorescein must be protected as an alkyl ester.
• the amide group can be present as an N-Fmoc (fluorenylmethoxycarbonyl) protected compound.
• Hydroxy groups can be protected from side reactions by acylation or silylation (t-butyldimethylsilyl).
• Amino groups can also be protected with trifluoroacetyl.
• the conjugates may be so unstable that they would already be destroyed under the conditions of deprotection in oligonucleotide synthesis.
• the synthesized oligonucleotide derivatives are characterized by Electro-spray ionization mass spectrometry (Stults and Masters, Rapid Commun. Mass. Spectr. 5 (1991) 350).
• oligonucleotide analogs of the formula IA according to the invention were based on tested their stability in serum and against known exonucleases.
• oligonucleotide analogs of formula IA are also stable to Snake venom phosphodiesterase. Unmodified oligonucleotides are made by Snake venom phosphodiesterase from the 3 'end and spleen phosphodiesterase degraded exonucleolytically from the 5 'end.
• oligonucleotide analogs of formula IA form Watsoh-Crick base pairing with complementary single-stranded Nucleotide sequences of stable double-stranded hybrids or by Watson-Crick and Hoogsteen base pairing stable triplex structures while using double-stranded nucleic acids via Hoogsteen base pairing tripelhelicale Form structures, the spacers (5'5'S) and (3'3'S) changing strands enable.
• oligonucleotide analogs Formula IA can therefore be used for therapy or prophylaxis of virals Infections or cancer.
• sequence I in the form of the natural sequence, that is to say without 3'-derivatization and 5'5'-spacer, is ineffective against HSV-1 in cell culture, since it is subject to rapid degradation in the serum.
• 3'-derivatized oligonucleotide analogs of the formula IA for example the sequence IA-2 (example 4b), inhibit the multiplication of HSV-1.
• An oligonucleotide of formula IA-4 (example 4e) of sequence IV modified with psoralen at both 3 'ends recognizes the origin of replication (ori L ) of the HSV-1 genome and inhibits its replication by triplex formation.
• the antiviral effectiveness of the psoralen conjugates can be significantly increased by irradiation with UV light.
• the HSV-1 genome with its 160,000 bases naturally offers countless other target sequences of different efficiency to inhibit virus replication.
• the therapeutic principle can also be applied to any other viruses, bacteria or other pathogens. The only requirement for a transfer to other disease triggers is that one knows the genes of these pathogens that are essential for the life cycle.
• Sequence V is directed, for example, against the APP770 gene promoter, which is responsible for the expression of the precursor protein of the plaque-forming amyloid proteins in Alzheimer's disease.
• Sequence VI simulates the SP1 binding region of the adenovirus E1b as a sense oligonucleotide and inhibits the transcription of E1b as a 3'-modified oligonucleotide analog with a 3'5 'spacer of the formula IB-2.
• oncogenes are abl, new, myc, myb, ras, fos, mos, erbB, ets, jun, p53, src and rel.
• Probes for nucleic acids in particular for DNA from oligonucleotide analogs Formula IA offer compared to the known oligonucleotide derivatives with a 3'-hydroxy group on the one hand the advantage of increased nuclease stability, on the other hand, they also allow the inclusion of the same or different ones Marker molecules at both ends of the oligonucleotide.
• the advantage is that selectively different marker groups within an oligonucleotide Get stimulated (double marking).
• the bifunctional derivatization can also used to place a marker on one end and the other end to introduce an additional function (e.g. affinity label).
• To this Purpose can, for example, incorporate biotin at a 3 'end of the oligonucleotide that recognizes avidin or streptavidin, while at the other 3'-end an alkylamino linker attached an acridinium ester chemiluminescent marker can be.
• the penetration behavior of the invention Oligonucleotide analogs cheaper than the unmodified oligonucleotides, especially when lipophilic residues have been introduced.
• oligonucleotide analogs for example, also as aids in biotechnology and Molecular biology can be used.
• the invention further relates to pharmaceutical preparations that are effective Amount of one or more compounds of formula IA or their physiologically acceptable salts, optionally together with physiologically compatible auxiliaries and / or carriers and / or together with others known active ingredients, as well as a method for producing them Preparations, characterized in that the active ingredient, together with the carrier and possibly other auxiliaries, additives or active ingredients in a suitable Dosage form brings.
• the application is preferably intravenous, topical or intranasally.
• 10 nmol of the oligonucleotide to be examined become 20% in 450 ul fetal calf serum dissolved in RPMI medium and 50 ml bidistilled water and incubated at 37 ° C. Then immediately and after 1, 2, 4, 7 and 24 hours 10 ⁇ l Samples for gel electrophoresis or 20 ⁇ l samples for HPLC taken to Interruption of the reaction was mixed with 5 ⁇ l or 10 ⁇ l formamide and opened for 5 minutes 95 ° C heated. For gel electrophoresis, the samples are reduced to a 15% Polyacrylamide gel (2% BIS) applied and this at about 3000 volt hours developed. The bands are made visible by the silver coloring.
• 2% BIS Polyacrylamide gel
• the antiviral activity of the compounds according to the invention is in vitro Try tested.
• the compounds according to the invention are described in different dilutions to cell cultures of HeLa and Vero cells in Given microtiter plates. After 3 hours, the cultures with different Human pathogenic viruses (e.g. herpes viruses HSV-1, HSV-2, orthomyxoviruses Influenza A2, Picornaviruses Rhinovirus 2) infected. 48 to 72 h after infection the success of therapy based on the cytopathogenic effect microscopic and after Neutral red image (color test after Finter) determined photometrically (Finter, N.B., In "Interferones", N.B. Finter et al., North Holland Publishing Co., Amsterdam, 1966). The minimum concentration at which about half of the infected cells do not show cytopathogenic effect is called minimal inhibitory concentration (MIC) considered.
• MIC minimal inhibitory concentration

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